Method for Suppressing Intermolecular Nonspecific Interaction and for Intensifying Intermolecular Specific Interaction on Metal Surface

ABSTRACT

The present invention provides a method of searching for a target molecule for a ligand immobilized on a metal surface or analyzing the interaction between a ligand and a target molecule, characterized in that the immobilization of the ligand on the metal surface via a hydrophilic spacers, and the method eliminates or suppresses a nonspecific interaction that prevents analysis of intermolecular interaction on a metal surface, and can intensify specific intermolecular interactions.

TECHNICAL FIELD

The present invention relates to a basic technology in intermolecularinteractions using a solid phase carrier. More specifically, the presentinvention relates to a technology to immobilize a molecule to beanalyzed onto a metal surface, and to measure and analyze theintermolecular interaction on the metal surface by making use of theinteraction, whereby a molecule exhibiting a specific interaction withthe molecule to be analyzed is selected and purified or the specificinteraction between the molecules is analyzed.

BACKGROUND ART

In recent years, attempts to search a molecule that exhibits a specificinteraction with a particular molecule using a technique based onintermolecular interactions, or research to investigate intermolecularinteractions in detail, has been actively conducted. This isspecifically represented by research wherein one molecule of thecombination of low molecule-low molecule, low molecule-high molecule, orhigh molecule-high molecule is immobilized onto a solid phase carrierand the interaction between the two molecules is measured, or researchwherein a desired target molecule (a molecule that exhibits a specificinteraction with a molecule immobilized onto a solid phase carrier) ispurified on the basis thereof. As examples of various techniques basedon intermolecular interactions, 1) target research using an affinityresin for the latter case, and 2) a method that applies surface plasmonresonance (Surface Plasmon Resonance: SPR) for the former case, areknown well. As examples of 1), the discovery of FKBP (FK506 bindingproteins), which is the immunosuppressant FK506-binding proteins, usingan affinity resin by Professor Schreiber in 1989 (discovery of FKBP12 asa protein that binds to FK506 in cells; for example, Nature (UK), vol.341, p 758-760, Oct. 26, 1989), the subsequently done discovery ofcalcineurin inhibitory action in the pharmacological action mechanism ofFK506 by an FK506—FKBP complex (for example, Cell (USA), vol. 66, No. 4p 807-815, Aug. 23, 1991), the discovery of HDAC as a target protein forthe anticancer agent Trapoxin (for example, Science (USA), vol. 272, p408-411, Apr. 19, 1996) and the like are known well. Also, as an exampleof 2), BIACORE (trade name), which employs a gold foil as a solid phasecarrier and enables an extensive investigation of an interaction betweena compound or a protein and the like and a protein and the like thatspecifically interacts therewith, is known well.

However, to date, in the above-described techniques, the presence of anonspecific intermolecular interaction that hampers the selection andpurification of a desired molecule based on a specific intermolecularinteraction has been posing such problems as 1) in target search usingan affinity resin, a nonspecific protein that masks a specific proteinduring analysis of a protein bound to the affinity resin using SDS geland the like exists and makes the detection of the specific proteindifficult, or 2) in analysis using BIACORE and the like, the presence ofa major peak resulting from nonspecific protein adsorption makes thedistinguishing of a peak due to specific protein binding difficult.Although these problems have empirically been considered as being causedby the solid phase carrier, which is an important basic technology,specifically by a surface property of the solid phase carrier, itremains yet to be known clearly which property is the causal factor fora nonspecific interaction, and how to efficiently suppress such anonspecific intermolecular interaction. For example, some resins such asTentaGel (Fluka Company, Cat. No=86364) have a PEG spacer, arechemically and physically stable, and are also used as resins foraffinity chromatography (e.g., Thorpe DS, Walle S., Combinatorialchemistry defines general properties of linkers for the optimal displayof peptide ligands for binding soluble protein targets to TentaGelmicroscopic beads., Biochem Biophys Res Commun 2000 Mar. 16;269(2):591-5), but basic technologies such as those concerning theidentity of the structure that contributes to suppression of anonspecific interaction and the way of the contribution remain unknown,and there is no sufficient information on to which extent thenonspecific interaction is suppressed or whether or not these resinsserve well as affinity resins. As such PEG spacers, TentaGel, which isdescribed above, and ArgoGel (Argonaut Company) are commerciallyavailable. Their structures are as follows

Also, resins comprising a sugar derivative having hydrophilic nature(e.g., AffiGel (AffiGel; Bio-Rad Company, Cat. No=153-2401), a Sepharosederivative (Pharmacia Company, ECH Sepharose 4B, Cat. No=17-0571-01) andthe like are known) exhibit minor nonspecific intermolecularinteractions, but they are physically and chemically unstable because oftheir identity as sugar derivatives and their use is limited.

If it is possible to artificially suppress nonspecific interactions inthe above-described techniques based on intermolecular interactions, itis considered that the necessity of determining whether the resultsobtained are due to specific protein binding or nonspecific proteinadsorption will be obviated, the frequency of research interruption dueto the substantial inability to differentiate both thereof willdecrease, and the consumption of protein and the like used will besignificantly reduced, so that significant cost reductions in terms oftime and labor, and the like will increase the applicability of thesetechniques.

The present inventors have already found heretofore that the presence ofa hydrophilic spacer for immobilization of a ligand onto a solid phasecarrier such as a resin and the like reduces a nonspecific interactionbetween the immobilized ligand molecule and/or the resin per se, and amolecule not specific to the ligand (WO2004/025297). However, suchmethod is based on the mechanism of improving an S/N ratio by mainlysuppressing a nonspecific intermolecular interaction, and there still isa demand for a method for intensifying the specific intermolecularinteraction itself.

The present invention aims at provision of a method for eliminating orsuppressing a nonspecific interaction that prevents analysis ofintermolecular interaction particularly on a metal surface, and furtheraims at provision of a method of purifying or analyzing a targetmolecule that specifically interacts with a ligand immobilized on ametal surface, while utilizing the method.

DISCLOSURE OF THE INVENTION

In view of the above-mentioned object, the present inventors haveconducted various studies and surprisingly found that introduction of ahydrophilic spacer into a solid phase carrier not only affords an actionto suppress a nonspecific interaction but also enhances a specificinteraction, particularly when a metal is used as a solid phase carrier.Furthermore, they have succeeded in more accurately searching a targetof a ligand, analyzing a specific interaction between a ligand and atarget molecule and the like, which resulted in the completion of thepresent invention.

That is, the present invention is as follows.

[1] A method of suppressing a nonspecific interaction between a ligandand/or a metal surface and a molecule other than a target molecule,which comprises, in a process of immobilizing the ligand onto the metalsurface and analyzing a specific interaction on the metal surfacebetween the ligand and the target molecule thereof, a treatment toreduce the hydrophobic property of the metal surface.[2] A method of intensifying a specific interaction between a ligand anda target molecule, which comprises, in a process of immobilizing theligand onto a metal surface and analyzing a specific interaction on themetal surface between the ligand and the target molecule of the ligand,a treatment to reduce the hydrophobic property of the metal surface.[3] A method of suppressing a nonspecific interaction between a ligandand/or a metal surface and a molecule other than a target molecule andintensifying a specific interaction between the ligand and the targetmolecule, which comprises, in a process of immobilizing the ligand ontothe metal surface and analyzing a specific interaction on the metalsurface between the ligand and the target molecule of the ligand, atreatment to reduce the hydrophobic property of the metal surface.[4] A method of suppressing a nonspecific interaction between a ligandand/or a metal surface and a molecule other than a target molecule,which comprises, in a process of immobilizing the ligand onto the metalsurface and selecting a target molecule using a specific interaction onthe metal surface between the ligand and the target molecule thereof, atreatment to reduce the hydrophobic property of the metal surface.[5] A method of intensifying a specific interaction between a ligand anda target molecule, which comprises, in a process of immobilizing theligand onto the metal surface and selecting the target molecule using aspecific interaction on the metal surface between the ligand and thetarget molecule thereof, a treatment to reduce the hydrophobic propertyof the metal surface.[6] A method of suppressing a nonspecific interaction between a ligandand/or a metal surface and a molecule other than a target molecule andintensifying a specific interaction between the ligand and the targetmolecule, which comprises, in a process of immobilizing the ligand ontothe metal surface and selecting the target molecule using a specificinteraction on the metal surface between the ligand and the targetmolecule thereof, a treatment to reduce the hydrophobic property of themetal surface.[7] The method of any one of [1]-[6] above, wherein the treatment toreduce the hydrophobic property of the metal surface is to introduce, atthe time of immobilization of the ligand onto the metal surfacer ahydrophilic spacer therebetween.[8] The method of [7] above, wherein the hydrophilic spacer has at leastany of the following characteristics while in a state bound to the metalsurface and the ligand:(i) the number of hydrogen bond acceptor is 6 or more,(ii) the number of hydrogen bond donor is 5 or more,(iii) the total number of hydrogen bond acceptor and hydrogen bond donoris 9 or more.[9] The method of [8] above, wherein said hydrophilic spacer further hasone or more carbonyl groups in the molecule thereof.[10] The method of [8] or [9] above, further characterized in that saidhydrophilic spacer does not have a functional group that becomespositively or negatively charged in an aqueous solution.[11] A method of immobilizing a ligand onto a metal surface andanalyzing a specific interaction on the metal surface between so theligand and a target molecule thereof, which comprises suppressing anonspecific interaction between the ligand and/or the metal surface anda molecule other than the target molecule by a treatment to reduce thehydrophobic property of the metal surface.[12] A method of immobilizing a ligand onto a metal surface andanalyzing a specific interaction on the metal surface between the ligandand a target molecule thereof, which comprises intensifying a specificinteraction between the ligand and the target molecule by a treatment toreduce the hydrophobic property of the metal surface.[13] A method of immobilizing a ligand onto a metal surface andanalyzing a specific interaction on the metal surface between the ligandand a target molecule thereof, which comprises suppressing a nonspecificinteraction between the ligand and/or the metal surface and a moleculeother than the target molecule and intensifying a specific interactionbetween the ligand and the target molecule, by a treatment to reduce thehydrophobic property of the metal surface.[14] A method of immobilizing a ligand onto a metal surfacer andselecting a target molecule using a specific interaction on the metalsurface between the ligand and the target molecule thereof, whichcomprises suppressing a nonspecific interaction between the ligandand/or the metal surface and a molecule other than the target moleculeby a treatment to reduce the hydrophobic property of the metal surface.[15] A method of immobilizing a ligand onto a metal surface, andselecting a target molecule using a specific interaction on the metalsurface between the ligand and the target molecule thereof, whichcomprises intensifying a specific interaction between the ligand and thetarget molecule by a treatment to reduce the hydrophobic property of themetal surface.[16] A method of immobilizing a ligand onto a metal surface, andselecting a target molecule using a specific interaction on the metalsurface between the ligand and the target molecule thereof, whichcomprises suppressing a nonspecific interaction between the ligandand/or the metal surface and a molecule other than the target moleculeand intensifying a specific interaction between the ligand and thetarget molecule, by a treatment to reduce the hydrophobic property ofthe metal surface.[17] The method of any one of [11]-[16] above, wherein the treatment toreduce the hydrophobic property of the metal surface is to introduce, atthe time of immobilization of the ligand onto the metal surface, ahydrophilic spacer therebetween.[18] The method of [17] above, wherein the hydrophilic spacer has atleast any of the following characteristics while in a state bound to themetal surface and the ligand:(i) the number of hydrogen bond acceptor is 6 or more,(ii) the number of hydrogen bond donor is 5 or more,(iii) the total number of hydrogen bond acceptor and hydrogen bond donoris 9 or more.[19] The method of [18] above, wherein said hydrophilic spacer furtherhas one or more carbonyl groups in the molecule thereof.[20] The method of [18] or [19] above, further characterized in thatsaid hydrophilic spacer does not have a functional group that becomespositively or negatively charged in an aqueous solution.[21] A screening method for a target molecule having a specificinteraction with a ligand, comprising at least the following steps:(i) immobilizing the ligand onto a metal surface via a hydrophilicspacer,(ii) contacting a sample that contains or does not contain the targetmolecule with the metal surface with the ligand immobilized thereonobtained in (i) above,(iii) identifying and analyzing a molecule that has exhibited or has notexhibited a specific interaction with the ligand, and(iv) judging a molecule that exhibits a specific interaction with theligand as the target molecule on the basis of the analytical resultsobtained in (iii) above.[22] The method of [21] above, wherein the hydrophilic spacer has atleast any of the following characteristics while in a state bound to themetal surface and the ligand:(i) the number of hydrogen bond acceptor is 6 or more,(ii) the number of hydrogen bond donor is 5 or more,(iii) the total number of hydrogen bond acceptor and hydrogen bond donoris 9 or more.[23] The method of [22] above, wherein said hydrophilic spacer furtherhas one or more carbonyl groups in the molecule thereof.[24] The method of [22] or [23] above, further characterized in thatsaid hydrophilic spacer does not have a functional group that becomespositively or negatively charged in an aqueous solution.[25] The method of any one of [7]-[10], [17]-[24] above, wherein thehydrophilic spacer has at least one partial structure represented by anyone formula selected from the group consisting of Formulas (Ia)-(Ie)below:

(In Formula (Ia),

A is an appropriate joining group,X₁-X₃ are the same or different and each is a single bond or a methylenegroup that may be substituted by a linear or branched alkyl group having1-3 carbon atoms,R₁-R₇ are the same or different and each is a hydrogen atom, a linear orbranched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup,m is an integer of 0-2, m′ is an integer of 0-10, m″ is an integer of0-2,when a plurality of R₃-R₇ units exist, they may be the same ordifferent, when a plurality of X₃ units exist, they may be are the sameor different;

in Formula (Ib),

n and n′ are the same or different and each is an integer of 1-1000;

in Formula (Ic),

p, p′ and p″ are the same or different and each is an integer of 1-1000;

in Formula (Id),

X₄ is a single bond or a methylene group that may be substituted by alinear or branched alkyl group having 1-3 carbon atoms,R₈-R₁₀ are the same or different and each is a hydrogen atom, a linearor branched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup,q is an integer of 1-7,when a plurality of R₈ units exist, they may be the same or different,when a plurality of X₄ units exist, they may be the same or different;

in Formula (Ie),

R₁₁-R₁₆ are the same or different and each is a hydrogen atom, a linearor branched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup,r is an integer of 1-10, r′ is an integer of 1-50,when a plurality of R₁₁-R₁₆ units exist, they may be the same ordifferent).[26] The method of [25] above, wherein the hydrophilic spacer has two ormore partial structures represented by any one formula selected from thegroup consisting of Formulas (Ia)-(Ie).[27] A solid phase carrier with a ligand immobilized thereon, which is ametal and has a hydrophilic spacer between the metal and the ligand.[28] The solid phase carrier of [27] above, wherein the hydrophilicspacer has at least one partial structure represented by any one formulaselected from the group consisting of Formulas (Ia)-(Ie).[29] The solid phase carrier of [27] or [28] above, wherein the metal isgold.[30] A method of confirming introduction of a hydrophilic spacer betweena ligand and a metal surface, which comprises, in a step of introducingthe hydrophilic spacer between them during immobilization of the ligandonto the metal surface, detecting a leaving group produced byelimination of a protecting group derived from the hydrophilic spacer.[31] The method of [30] above, wherein the leaving group is detected bymass analysis.[32] The method of [30] above, wherein the protecting group is a9-fluorenylmethyloxycarbonyl group.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of the electrophoresis showing the comparisonresults between the case wherein a ligand is immobilized onto a goldfilm surface via a hydrophilic spacer (Production Example 11) and thecase wherein a ligand is immobilized onto a gold film surface without ahydrophilic spacer (Reference Example 1), with respect to the adsorptionof a nonspecific binding protein to the gold film surface (ligand) andbinding thereto of a specific protein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the finding that nonspecificintermolecular interactions (e.g., represented by nonspecific adsorptionof a protein to a solid phase carrier), which have been viewed as beingproblematic in the technology to analyze and utilize a specificintermolecular interaction, are due to the hydrophobic interactionbetween the solid phase surface in a solid phase carrier and a moleculesuch as of a protein. Particularly, the present invention also providesa method of suppressing nonspecific adsorption of various molecules to ametal surface and increasing the amount of specific adsorption, by atreatment to reduce the hydrophobic property of the metal surface.

In the present specification, the hydrophobic property can generally berepresented by a hydrophobicity parameter, and can, for example, berepresented by partition coefficient, specifically by LOGP. Incalculating LOGP, CLOGP (a predicted value obtained using a softwareprogram for estimating hydrophobicity parameters of a compound by meansof a computer; can be calculated using, for example, Corwin/Leo'sprogram (CLOGP, Daylight Chemical Information System Co., Ltd)) and thelike are conveniently utilized, but the hydrophobicity parameter is notlimited to CLOGP. In the present invention, as the tendency forhydrophobicity increases qualitatively, nonspecific interactionsincrease. For example, referring to CLOGP, the greater the CLOGP is, thehigher the hydrophobicity is; the increase in CLOGP correlates to theincrease in a nonspecific interaction (e.g., nonspecific adsorption of aprotein to a metal surface). Here, a change in a hydrophobicityparameter can, for example, be performed by changing the ligand to beimmobilized onto the metal surface to one having various values (e.g.,CLOGP) of hydrophobicity parameter, and it is also possible to moderateor reduce the hydrophobic property of the metal surface by introducing ahydrophilic spacer between the metal surface and the ligand.

Introducing the spacer is a preferred embodiment in cases wherein it isnecessary to immobilize onto a metal surface a ligand expected to have ahigh CLOGP value; a case wherein a hydrophilic spacer is used as a meansof suppressing a nonspecific intermolecular interaction is hereinafterdescribed in detail.

The present invention provides a technology to analyze the interactionbetween a molecule immobilized onto a metal surface (in the presentspecification, also defined as ligand) and a molecule that exhibits aspecific interaction with the molecule described above (in the presentspecification, also defined as target molecule), and a technology toidentify and select the target molecule on the basis of such analysis.In the present specification, the terms ligand and target molecule areintended to mean a combination of members that exhibit a specificintermolecular interaction with each other, and their designations arevariable depending on which member of the combination to immobilize asthe ligand onto the solid phase and leave the other member as the targetmolecule, that is, which member to immobilize onto the solid phase.There can be more than one kind of the target molecule that exhibits aspecific interaction with the ligand, and likewise there can be morethan one kind of the ligand that exhibits a specific interaction withthe target molecule. In the present specification, the terms ligand andtarget molecule do not refer to particular molecules but are intended tomean individual molecules that exhibit a specific interaction with eachother.

A “specific interaction” is a characteristic action to specificallyrecognize, and bind to, a particular ligand (a particular targetmolecule) only; the relation of a specific receptor to an agonist or anantagonist, the relation of an enzyme to a substrate, and, for example,the relation of an FK506-binding protein (target molecule) to FK506(ligand), the relation of a steroid hormone receptor to a steroidhormone (e.g., dexamethasone and glucocorticoid receptor), the relationof HDAC to the anticancer agent trapoxin, and the like apply to a“specific interaction”. On the other hand, a “nonspecific interaction”refers to an action the subjects of binding by which encompass a broadrange of molecules and are not limited to particular molecules, andwhich produces a situation that is variously changeable depending onreaction conditions; in the present invention, this term means anunparticular intermolecular action to bind or adsorb to the ligand on asolid phase or the solid phase carrier surface. A “nonspecificinteraction” is risky in that the binding based on a “specificinteraction” is possibly overlooked as it hampers, or is confused with,the binding of the ligand and the target molecule based on a “specificinteraction”.

In the present invention, “to analyze a specific interaction” is toobtain the extent of the specific interaction between a ligand and atarget molecule as interaction information, which can, for example, beobtained as numerical values of Kd (dissociation rate constant), Ka(association rate constant) and the like. In the present invention,“selection” is intended to mean determining whether or not the moleculein question exhibits a specific interaction with the ligand on the basisof the above-described interaction information, and identifying thetarget molecule.

In the present invention, a treatment to reduce the hydrophobic propertyof the metal surface is essential. As an example of the treatment, amethod of introducing a hydrophilic spacer, at the time ofimmobilization of the ligand to the metal surface, therebetween, can bementioned. By introducing a hydrophilic spacer, the hydrophobic propertyof the metal surface is altered, so that a nonspecific interaction canbe suppressed, as well as a specific interaction can be intensified. Byusing such a means of suppressing a nonspecific interaction, called ahydrophilic spacer, introduced between the metal surface and the ligand,it is possible to identify and select a molecule (target molecule) thatexhibits a specific interaction with the ligand, and to accuratelymeasure the interaction therebetween.

The metal as a solid phase carrier to be used in the present inventionincludes various kinds generally used in this field, which may bespecifically gold, silver, iron, silicone and the like. These may haveany shape, which is appropriately determined according to the kind ofthe above-mentioned metal, the analysis to be performed thereafter ofthe interaction between a ligand and a target molecule, and a method tobe used for the identification and selection of the target molecule. Forexample, plates, thin films, threads, coils and the like can bementioned; metallic thin films can be preferably used as carriers forBIACORE and the like by surface plasmon resonance.

Although the metal used in the present invention, as described above, isnot subject to limitation as to the kind and form thereof, one having astructural hindrance such that the ligand is not immobilizable thereonor the ligand is immobilizable thereon but cannot exhibit a specificinteraction with the target molecule, of course, is undesirable forembodying the present invention because it complicates the operation dueto the necessity of an additional step or is unusable in some cases.

In the present invention, “hydrophilic spacer” refers to a substancethat is introduced to become a group that interlies between a metalsurface and a ligand at the time of immobilization of the ligand ontothe metal surface, and is hydrophilic. Degrees of hydrophilicity aredescribed below. Here, “a spacer interlies” means that the spacer ispresent between a functional group in the solid phase and a functionalgroup in the ligand. The spacer binds to the functional group in thesolid phase at one end and binds to the functional group in the ligandat the other end.

Also, the hydrophilic spacer may be one obtained by sequentially bindingand polymerizing 2 or more compounds, as long as it is capable ofeventually functioning as a group that interlies between the metalsurface and the ligand. Preferably, the hydrophilic spacer is obtainedby a polymerization reaction of a unit compound. The process to bind orpolymerize 2 or more compounds is preferably conducted on a metalsurface. Binding of the metal surface and the hydrophilic spacer,binding of the hydrophilic spacer and the ligand, and binding andpolymerization of individual components that constitute the hydrophilicspacer are based on a covalent bond or a non-covalent bond, such as anamide bond, a Schiff base, a C—C bond, an ester bond, a hydrogen bond ora hydrophobic interaction, all of which are formed using materials andreactions known in the art.

In the present invention, the hydrophilic spacer introduced between ametal surface and a ligand as a means of reducing the hydrophobicproperty of the metal surface is not subject to limitation, as long asit alters the hydrophobic property of the metal surface to eliminate orsuppress the nonspecific interaction and to intensify the specificinteraction, and is preferably a compound having the number of hydrogenbond acceptor (HBA; hydrogen bond acceptor) of 6 or more, or the numberof hydrogen bond donor (HBD; hydrogen bond donor) of 5 or more, or thetotal number of HBA and HBD per spacer molecule of 9 or more while in astate bound to metal surface and the ligand (a hydrophilic spacer inthis state is hereinafter referred to as “hydrophilic spacer moiety” forconvenience). Also, the hydrophilic spacer may be a compound that meetstwo or all of these conditions. Particularly preferably, the number ofHBA is 7 or more and the number of HBD is 6 or more.

Here, the number of hydrogen bond acceptor (the number of MBA) is thetotal number of nitrogen atoms (N) and oxygen atoms (O) contained, andthe number of hydrogen bond donor (the number of HBD) is the totalnumber of NH and OH contained (for example, Advanced Drug DeliveryReviews, Netherland, 23 (1997) p. 3-25).

For immobilization of a ligand on the metal surface, a method comprisingadsorbing a thiol compound or disulfide compound onto a metal surface toform a Self-Assembled Monolayer (SAM), and immobilizing the ligand onits terminal is generally employed (see Dojin News No. 91 p 3 (1999)).By forming a SAM on a metal surface and immobilizing the ligand on thesurface via a functional group in the SAM, an interaction between aligand and a target molecule can be detected by a gold-modifiedelectrode, surface plasmon resonance, Quartz Crystal Microbalance (QCM)and the like (specifically, detected based on the changes in electriccurrent, reflection angle or vibration frequency, respectively). Forexample, when gold is used as a metal to be the solid phase carrier,alkanethiol is used as a thiol compound.

In the present invention, therefore, a connection part minimum requiredfor binding a ligand to a metal surface (specifically, a part derivedfrom the above-mentioned thiol compound or disulfide compound) is notincluded in the hydrophilic spacer of the present invention, even if itis a group intervening between a metal surface and a ligand, andtherefore, is not included in the number of HBA and the number of HBD.To facilitate binding of a ligand to a hydrophilic spacer, moreover, itis possible to bind or introduce an optional group to or into theligand, in between the ligand and the hydrophilic spacer, before bindingto the hydrophilic spacer. However, since they are appropriatelyselected according to the ligand, and considered to less contributing tothe mitigation of the hydrophobic property of the metal surface, N, O,NH and OH contained in such group are not counted in the number of HBDand the number of HBA in the present invention. For introduction of theoptional group in between the ligand and the hydrophilic spacer, theaforementioned various covalent bonds and noncovalent bonds can beutilized, using materials and reactions known in this field.

Under the circumstances of the invention of the present application,nonspecific interactions cannot be fully suppressed and specificinteractions cannot be sufficiently intensified, unless at least one,preferably 2 or more, of the conditions of the number of HBA of 6 ormore (preferably 7 or more), the number of HBD of 5 or more (preferably6 or more), and the total number of HBA and HBD of 9 or more, are met.Therefore, in the hydrophilic spacer of the invention of the presentapplication, “hydrophilic” means that the above-described requirementsare met. In the present invention, the upper limit of the number of HBDor the number of HBA of the hydrophilic spacer is not subject tolimitation, as long as the spacer is hydrophilic and capable ofsuppressing a nonspecific interaction; by appropriately repeating apolymerization reaction and the like, a spacer having an extremely highhydrophilicity can be obtained. Also, the spacer may be a high-molecularsubstance such as a protein; from this viewpoint, the upper limit shouldbe at a value of 50,000 or so in all cases.

In the present invention, moreover, a hydrophilic spacer with aphysically or chemically unstable compound, such as a sugar derivativeor a sepharose derivative, as a basic skeleton, is not preferable foruse, even if it satisfies the degree of “hydrophilicity” defined above,since it may be too unstable to stand the immobilization of a ligand andsubsequent various treatments. Specifically, carboxymethyldextranconventionally used for immobilization of a ligand on a gold thin filmis not included in the hydrophilic spacer to be used in the presentinvention.

Furthermore, the hydrophilic spacer used in the present invention ispreferably one that does not exhibit a nonspecific interaction (e.g.,protein adsorption to the spacer and the like) per se. Specifically, itis preferable that the spacer does not have a functional group thatbecomes positively or negatively charged in an aqueous solution; as thefunctional group, an amino group (but excluding cases wherein afunctional group that attenuates the basicity of the amino group (e.g.,a carbonyl group, a sulfonyl group) is bound to the amino group), acarboxyl group, a sulfuric acid group, a nitric acid group, a hydroxamicacid group and the like can be mentioned. Here, “in an aqueous solution”specifically refers to an environment wherein the process to analyze theinteraction between a ligand and a target molecule on a metal surface,the process to select the target molecule, or a binding reaction (areaction based on a specific interaction) of the ligand and the targetmolecule performed to screen for the target molecule is conducted, andwhereunder the hydrophilic spacer ionizes when having a functional groupthat becomes positively or negatively charged. Such conditions are, forexample, in an aqueous solution, pH 1-11, temperature 0° C.-100° C.,preferably pH nearly neutral (pH 6-8), about 4° C. to about 40° C. orso.

Furthermore, the hydrophilic spacer used in the present inventionpreferably has 1 or more carbonyl groups in the molecule thereof, asunderstood from the various structures or compounds described below aspreferable examples of the hydrophilic spacer.

For example, the hydrophilic spacer used in present invention is acompound that has at least one partial structure represented by any oneformula selected from the group consisting of Formulas (Ia)-(Ie) below.

(In Formula (Ia),

A is an appropriate joining group,X₁-X₃ are the same or different and each is a single bond or a methylenegroup that may be substituted by a linear or branched alkyl group having1-3 carbon atoms,R₁-R₇ are the same or different and each is a hydrogen atom, a linear orbranched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup,m is an integer of 0-2, m′ is an integer of 0-10, m″ is an integer of0-2,when a plurality of R₃-R₇ units exist, they may be the same ordifferent, when a plurality of X₃ units exist, they may be the same ordifferent;

in Formula (Ib),

n and n′ are the same or different and each is an integer of 1-1000;

in Formula (Ic),

p, p′ and p″ are the same or different and each is an integer of 1-1000;

in Formula (Id),

X₄ is a single bond or a methylene group that may be substituted by alinear or branched alkyl group having 1-3 carbon atoms,R₈-R₁₀ are the same or different and each is a hydrogen atom, a linearor branched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup,q is an integer of 1-7,when a plurality of R₈ units exist, they may be the same or different,when a plurality of X₄ units exist, they may be the same or different;

in Formula (Ie),

R₁₁-R₁₆ are the same or different and each is a hydrogen atom, a linearor branched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup,r is an integer of 1-10, r′ is an integer of 1-50,when a plurality of R₁₁-R₁₆ units exist, they may be the same ordifferent).

In the present specification, referring to the definitions forindividual groups, the “appropriate joining group” is not subject tolimitation, as long as it is capable of joining mutually adjoiningsites, and specifically the following groups are used.

(in the formulas, R₁₇ is a hydrogen atom or a linear or branched alkylgroup having 1-3 carbon atoms, R₁-R₂, are the same or different and eachis a hydrogen atom, a linear or branched alkyl group having 1-3 carbonatoms, —CH₂OH or a hydroxyl group, R₂₂-R₂₆ are the same or different andeach is a hydrogen atom or a linear or branched alkyl group having 1-3carbon atoms (the alkyl group may be substituted by a hydrophilicsubstituent such as a hydroxyl group, a carboxylic acid group, or anamino group))

In the present specification, referring to the definitions forindividual groups, as examples of the “linear or branched alkyl grouphaving 1-3 carbon atoms”, a methyl is group, an ethyl group, a propylgroup, an isopropyl group and the like can be mentioned.

In the present specification, “a methylene group that may be substitutedby a linear or branched alkyl group having 1-3 carbon atoms”, isintended to mean an unsubstituted methylene group and a methylene groupsubstituted by 1 or 2 of the above-described linear or branched alkylgroups having 1-3 carbon atoms.

The hydrophilic spacer of the present invention may have two or more ofthe above-described partial structure; in that case, the partialstructures may be represented by the same formula or represented bydifferent formulas.

At least one kind of the above-described hydrophilic spacer isimmobilized onto a metal surface. The number of spacers on the metalsurface is not subject to limitation, and can be appropriatelydetermined by those skilled in the art according to the kind and amountof the ligand, the kind and amount of the target molecule, and the kindand characteristic of the spacer used, and needs not be determined,provided that the desired intermolecular interaction can be detected.Usually, the spacer is immobilized using an excess amount thereofrelative to the metal used as a solid phase carrier and is the ligand.The hydrophilic spacers that have not bound to the metal surface caneasily be removed from the reaction system by a treatment such aswashing.

In the present invention, the ligand to be immobilized onto a metalsurface is not subject to limitation, and may be a known compound or anovel compound that will be developed in the future. Also, the ligandmay be a low molecular compound or a high molecular compound. Here, alow molecular compound refers to a compound having a molecular weight ofless than 1000 or so; for example, an organic compound commonly usableas a pharmaceutical, a derivative thereof, and an inorganic compound canbe mentioned; specifically, a compound produced by making use of amethod of organic synthesis and the like, a derivative thereof, anaturally occurring compound, a derivative thereof, a small nucleic acidmolecule such as a promoter, various metals, and the like can bementioned; and desirably, an organic compound that can be used as apharmaceutical, a derivative thereof, or a nucleic acid molecule can bereferred to. Also, as the high molecular compound, a compound having amolecular weight of 1000 or more or so, which is a protein, apolynucleic acid, a polysaccharide, or a combination thereof, and thelike can be mentioned, and a protein is desirable. These low molecularcompounds or high molecular compounds are commercially available if theyare known compounds, or can be obtained via steps such as of collection,production and purification according to various publications. These maybe of natural origin, or may be prepared by gene engineering, or may beobtained by semi-synthesis and the like.

In the present invention, a process to select a target molecule on thebasis of the specific interaction with the above-described ligand on ametal surface having the ligand immobilized thereon is necessary.Therefore, the target molecule is not subject to limitation, as long asit specifically interacts with the ligand, and is expected to be a knowncompound in some cases or a novel substance in other cases. The targetmolecule may be a low molecular compound or a high molecular compound.When the target molecule is a low molecular compound, the targetmolecule can be selected on the basis of the specific interaction withthe ligand that is a low molecular compound, which is a low molecularcompound-low molecular compound interaction, or on the basis of thespecific interaction with the ligand that is a high molecular compound,which is a high molecular compound-low molecular compound interaction.Also, when the target molecule is a high molecular compound, the targetmolecule can be selected on the basis of the specific interaction withthe ligand that is a low molecular compound, which is a low molecularcompound-high molecular compound interaction, or on the basis of thespecific interaction with the ligand that is a high molecular compound,which is a high molecular compound-high molecular compound interaction.A preferable combination of the ligand and the target molecule is thecombination of a low molecular compound and a high molecular compound,or the combination of a high molecular compound and a high molecularcompound.

Analysis of the interaction of the ligand with the target molecule andselection of the target molecule are conveniently conducted on a metalsurface which is a solid phase. When a candidate substance isanticipated as the target molecule, it is possible to bring thecandidate substance alone into contact with the above-described ligandimmobilized on the metal surface, measure the interaction therebetween,and determine whether or not the candidate substance is the targetmolecule; usually, by bringing a sample that contains a plurality ofsubstances (a high molecular compound and/or a low molecular compound)into contact with the ligand, and measuring the presence or absence ofan interaction between each of the plurality of substances (the highmolecular compound and/or the low molecular compound) and the ligand andthe extent of the interaction, whether or not each substance is thetarget molecule is determined and the target molecule is selected. Here,the sample that contains a plurality of substances may consistessentially of known compounds, may contain some novel compounds, andmay consist essentially of novel compounds. However, according to searchof target molecules for ligands, or recent advances in proteomeanalysis, it is desirable that the sample be a mixture essentially ofcompounds of known structures. As the sample consisting essentially ofknown compounds, a mixture of proteins prepared by gene engineeringusing Escherichia coli and the like, and the like can be mentioned; asthe sample that contains some novel compounds, a cell or tissue extract(Lysate) can be mentioned; as the sample that consists essentially ofnovel compounds, a mixture of novel proteins whose functions andstructures are yet unknown, or newly synthesized compounds and the like,can be mentioned. When the sample is a mixture, especially when itcontains known compounds, the contents of these compounds in the samplemay optionally be set at desired levels in advance, From the viewpointof searching a target molecule for a ligand, target molecule to beselected is preferably a low molecular compound or a high molecularcompound, and for searching a target molecule in the body of an animalsuch as a human, the target molecule is preferably a high molecularcompound.

The present invention provides, using the above-described ligandimmobilized on the metal surface, a method of screening for a targetmolecule that exhibits a specific interaction with the ligand. Thescreening method includes at least the following steps. Note that therespective definitions for the ligand, the target molecule, the metal(metal surface), and the hydrophilic spacer in this screening method areas described above.

(1) A Step of Immobilizing the Ligand onto a Metal Surface Via aHydrophilic Spacer.

This step comprises binding of the ligand and the hydrophilic spacer andbinding of the hydrophilic spacer and the metal surface. It is possibleto bind the hydrophilic spacer to the ligand and then bind the complexthereof to the metal surface, and also possible to bind the ligand afterthe hydrophilic spacer is bound to the metal surface; whether or not theligand has been immobilized onto the metal surface can be confirmed byutilizing a reaction based on a particular structure or substituent andthe like contained in the ligand or an optionally chosen group that hasbeen bound and introduced to the ligand in advance, and the like. Forexample, a method comprising detecting or measuring a leaving groupproduced during elimination of a 9-fluorenylmethyloxycarbonyl group(Fmoc group), which is an amino-protecting group in a ligand orhydrophilic spacer, and the like can be employed. Each binding isperformed by utilizing a reaction in common use in the art. As aconvenient and accurate means, a method utilizing an amide bondformation reaction can be mentioned. This reaction can, for example, beperformed according to “Pepuchido Gousei no Kiso to Jikken” (ISBN4-621-02962-2, Maruzen, first edition issued in 1985). Regarding thereagents and solvents used in each reaction, those in common use in therelevant field can be utilized, and are appropriately selected accordingto the binding reaction employed.

(2) A Step of Contacting a Sample that Contains or does not Contain theTarget Molecule with the Metal with the Ligand Immobilized ThereonObtained in (1) Above.

The sample used in this step is one containing a plurality of substancesas described above. The mode of embodiment thereof is not subject tolimitation, and can be appropriately changed according to the metal usedas solid phase carrier and their shape, principles, means and methods touse for the identification or analysis in the subsequent steps (3) and(4). For example, when analysis is performed by BIACORE (trade name) andusing a gold thin film on which a ligand has been immobilized, it ispreferable that the sample be liquid. In the case of a sample that doesnot contain the target molecule, identification and analysis of amolecule (a plurality of kinds present in some cases) that has notexhibited a specific interaction with the ligand in step (3) areconducted. In the case of a sample that contains the target molecule,the target molecule (a plurality of kinds present in some cases) thathas exhibited a specific interaction with the ligand in step (3) isidentified and analyzed. The method of bringing the sample and the metalsurface into contact with each other is not subject to limitation, aslong as the target molecule in the sample can bind to the ligandimmobilized on the metal surface, and can be appropriately changedaccording to the kinds of metal used and their shape, principles, meansand methods to use for the identification or analysis in the subsequentsteps (3) and (4). For example, when a gold thin film on which a ligandhas been immobilized is used, the treatment includes immersion of thegold thin film in a liquid sample and the like.

(3) A Step of Identifying and Analyzing a Molecule that has Exhibited orhas not Exhibited a Specific Interaction with the Ligand.

Although this step can be appropriately changed according to the kindsand shape of the metal used as solid phase carrier and the kinds of theligand, and the like, it is conducted by various methods in common usein the art to identify a low molecular compound or a high molecularcompound. Also, the step can also be performed by a method that will bedeveloped in the future. For example, when a gold thin film on which aligand has been immobilized is used as a metal having a ligandimmobilized on its surface [step (1)], the target molecule is bound tothe ligand by the subsequent addition of the sample [step (2)]. It isalso possible to dissociate the target molecule bound from the ligand bya treatment such as altering the polarity of the buffer solution orfurther adding the ligand in excess, and then identify the targetmolecule, or to extract the target molecule with a surfactant and thelike while remaining in a state bound to the ligand on the metalsurface, and then identify the target molecule. As the method ofidentification specifically, known techniques such as electrophoresis,immunoblotting and immunoprecipitation, which employ immunologicalreactions, chromatography, mass spectrometry, amino acid sequencing, NMR(especially for low-molecules), and reactions utilizing surface plasmonresonance, or combinations of these methods can be used. Although thestep of identifying a molecule that does not bind to the ligand can alsobe conducted in accordance with the above-described method ofidentifying a molecule that binds to the ligand, it is preferable that atreatment such as concentration or crude purification be conducted inadvance before entering the identification step, since a moleculecontained in the trough fraction from the column is the subject ofidentification. On the basis of the data obtained and existing reports,each molecule is identified, and whether or not it is a target moleculefor the ligand is determined.

Also, this step may be automated. For example, it is also possible todirectly read data on various molecules, which have been obtained bytwo-dimensional electrophoresis, and identify the molecules on the basisof existing databases.

A general method of producing the hydrophilic spacer of the presentinvention is described below, but it is obvious to those skilled in theart that the same can also be produced by other methods in common use inthe art or combinations thereof.

Note that the abbreviations used in the present specification are asfollows.

ABBREVIATION FORMAL DESIGNATION

-   Ac Acetyl group-   Bn Benzyl group-   Bu₃P Tributylphosphine-   DMAP Dimethylaminopyridine-   DMF Dimethylformamide-   EDC 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide-   Et Ethyl group-   Fmoc 9-Fluorenylmethyloxycarbonyl group-   Fmoc-OSu 9-Fluorenylmethylsuccinimidylcarbonate-   Gold foil Gold film-   HOBt 1-Hydroxybenzotriazole-   HyT Hydrazinotartaric amide-   Me Methyl group-   PEG Polyethylene glycol-   Ph₃P Triphenylphosphine-   PyBOP Benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium    hexafluorophosphate-   TBAF Tetrabutylammonium fluoride-   TBDMS tert-Butyldimethylsilyl group-   TBDMSOTf Trifluoromethanesulfonic acid t-butyldimethylsilyl group-   TBDPS tert-Butyldiphenylsilyl group-   TBS tert-Butyldimethylsilyl group-   tBu tert-Butyl group-   TFA Trifluoroacetic acid-   THF Tetrahydrofuran-   TMAD N,N,N′,N′-tetramethylazodicarboxamide-   Tr Trityl group-   Ts Tosyl group (toluenesulfonyl group)-   WSC Water-soluble carbodiimide    (N-ethyl-N′-(3′-dimethylaminopropyl)carbodiimide)

Process 1: Production Method (1) for the Hydrophilic Spacer Having aPartial Structure Represented by General Formula (Ia)

(m=1, m′=2, m″=1)

In the formulas, W₁-W₄ are hydroxyl-group-protecting groups, Z₁ is acarboxyl-group-protecting group, and Y₁ is an amino-group-protectinggroup. X_(3′), has the same definition as X₃ and X_(3″) has the samedefinition as X₃. R_(5′), has the same definition as R₅ and R_(5″) hasthe same definition as R₅. Also, the definitions for the otherindividual symbols are as described above.

As the hydroxyl-group-protecting group, an optionally chosen group incommon use in the art is used; specifically, alkyl groups such as atert-butyl group; acyl groups such as an acetyl group, a propionylgroup, a pivaloyl group and a benzoyl group; alkoxycarbonyl groups suchas a methoxycarbonyl group and a tert-butoxycarbonyl group;aralkyloxycarbonyl groups such as a benzyloxycarbonyl group; arylmethylgroups such as a benzyl group and a naphthylmethyl group; silyl groupssuch as a trimethylsilyl group, a triethylsilyl group, atert-butyldimethylsilyl group and a tert-butyldiphenylsilyl group; loweralkoxymethyl groups such as an ethoxymethyl group and a methoxymethylgroup, and the like can be mentioned as examples, and preferably, atert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, amethoxymethyl group and a tert-butyl group can be mentioned. As thecarboxyl-group-protecting group, an optionally chosen group in commonuse in the art is used; specifically, linear or branched lower alkylgroups having 1-6 carbon atoms, such as a methyl group, an ethyl group,a propyl group, a tert-butyl group, an isobutyl group and an allylgroup; aralkyl groups such as a benzyl group; silyl groups such as atert-butyldimethylsilyl group and a tert-butyldiphenylsilyl group, andthe like can be mentioned as examples; preferably, an allyl group, atert-butyl group, a benzyl group and a tert-butyldiphenylsilyl group canbe mentioned. As the amino-group-protecting group, an optionally chosengroup in common use in the art is used; specifically, loweralkoxycarbonyl groups such as a tert-butoxycarbonyl group, amethoxycarbonyl group and 9-fluorenylmethyloxycarbony group;aralkyloxycarbonyl groups such as a benzyloxycarbonyl group; aralkylgroups such as a benzyl group; substituted sulfonyl groups such as abenzenesulfonyl group, a p-toluenesulfonyl group and a methanesulfonylgroup, and the like can be mentioned as examples, preferably, atert-butoxycarbonyl group and a benzyloxycarbonyl group can bementioned.

Amino group protection and deprotection, carboxyl group protection anddeprotection, and hydroxyl group deprotection are appropriatelyperformed using known methods and reagents according to the protectivegroup used. In addition, when a compound has plural“amino-group-protecting groups”, “carboxyl-group-protecting groups”and/or “hydroxyl-group-protecting groups”, they may be the same ordifferent and are appropriately selected according to the moieties inneed of protection.

The reaction to dehydration-condense compound (a-4) and compound (a-2)by amidation is normally conducted in the presence of equivalent amountsof the amino compound and the carboxylic acid, using 1.1 equivalents orso of a condensing agent such as N-ethyl-N′-dimethylaminocarbodiimide orN-hydroxy-benzotriazol, in a solvent such as DMF or methylene chlorideat room temperature for 1 hour to 10 hours or so.

Process 2: Production Method (2) for the Hydrophilic Spacer Having aPartial Structure Represented by General Formula (Ia)

(m=2, m′=0, m″=2)

In the formulas, Y₂ is an amino-group-protecting group. R_(3′), has thesame definition as R₃ and R_(3″) has the same definition as R₃. R_(4′)has the same definition as R₄ and R_(4″) has the same definition as R₄.R_(6′) has the same definition as R₆ and R_(6″) has the same definitionas R₆. R_(7′) has the same definition as R₇ and R_(7″) has the samedefinition as R₇. The definitions for the other individual symbols areas described above. As examples of the amino-group-protecting group, thesame as those described above can be mentioned. Amino group deprotectionis appropriately performed using known methods and reagents according tothe protective group used.

The reaction to dehydration-condense compound (a-9) and compound (a-10)by amidation is normally conducted in the presence of equivalent amountsof the amino compound and the carboxylic acid, using 1.1 equivalents orso of a condensing agent such as N-ethyl-N′-dimethylaminocarbodiimide orN-hydroxy-benzotriazol, in a solvent such as DMF or methylene chlorideat room temperature for 1 hour to 10 hours or so.

Process 3: Production Method (3) for the Hydrophilic Spacer Having aPartial Structure Represented by General Formula (Ia)

(m=1, m′=0, m″=0)

In the formulas, Y₃ is an amino-group-protecting group, and thedefinitions for the other individual symbols are as described above. Asexamples of the amino-group-protecting group, the same as thosedescribed above can be mentioned.

The reaction to dehydration-condense compound (a-14) and compound (a-15)by amidation is normally conducted in the presence of equivalent amountsof the amino compound and the carboxylic acid, using 1.1 equivalents orso of a condensing agent such as N-ethyl-N′-dimethylaminocarbodiimide orN-hydroxy-benzotriazol, in a solvent such as DMF or methylene chlorideat room temperature for 1 hour to 10 hours or so.

Process 4: Production Method for the Hydrophilic Spacer Having a PartialStructure Represented by General Formula (Ib)

(n−1=n′−1=n₂)

In the formulas, W₅-W₇ are hydroxyl-group-protecting groups, Halrepresents a halogen atom (chlorine atom, bromine atom, iodine atom,fluorine atom), and the definitions for the other individual symbols areas described above. As examples of the hydroxyl-group-protecting group,the same as those described above can be mentioned. Note that n₂ is n−1or n′−1 (n and n′ are as described above).

Hydroxyl group protection and deprotection is appropriately performedusing known methods and reagents according to the protective group used.

The halogen substitution reaction of compound (b-4) to compound (b-5) isnormally conducted by reacting 2-3 equivalents of carbon tetrabromideand 1-2 equivalents of triphenylphosphine to 1 equivalent of the alcoholcompound in a solvent such as methylene chloride, at 0° C. to roomtemperature, is for 1 hour to several hours.

The dehydration-condensation reaction of compound (b-6) and compound(b-2) is normally conducted by reacting 1 equivalent of the alcoholcompound and 1 equivalent of tributylphosphine in a toluene solvent atroom temperature for 1 hour or so, adding thereto 1 equivalent of thephenol compound and a condensing agent such as1,1′-azobis(N,N-dimethylformamide), and allowing the reaction at 0-50°C. for several hours to overnight.

The condensation reaction of compound (b-8) and compound (b-5) isnormally conducted by reacting 1 equivalent of the phenol compound andabout 10 times equivalents of a strong base like sodium hydride inexcess at 0-10° C. in a solvent such as THF for 10-60 minutes or so,adding thereto 2 equivalents or so of the halogen compound, and allowingthe reaction at room temperature for 1-10 hours or so.

In the formulas, Alk is a linear or branched alkyl group having 1-3carbon atoms (defined as described above), Y₄ is anamino-group-protecting group, and the definitions for the otherindividual symbols are as described above. As examples of thehydroxyl-group-protecting group and the amino-group-protecting group,the same as those described above can be mentioned.

Hydroxyl group or amino group deprotection or carboxyl groupdeprotection is appropriately performed using known methods and reagentsaccording to the protective group used.

Alkoxycarbonylation of compound (b-10) to compound (b-11) is normallyconducted by reacting 1 equivalent of the alcohol compound and 3-5 timesequivalents or so of a strong base like sodium hydride in excess at0-10° C. in a solvent such as THF, for 10-60 minutes or so, addingthereto 3-5 times equivalents or so of the halogen compound (bromoaceticacid-tert-butyl ester) in excess, and allowing the reaction at roomtemperature for 1-10 hours or so.

Azidation of compound (b-12) to compound (b-13) is normally conducted byreacting 1 equivalent of the alcohol compound, 1.5 equivalents or so ofp-toluenesulfonyl chloride, and 0.2 equivalents or so of a base like4-dimethylaminopyridine in a solvent such as pyridine at 30-50° C. forseveral hours, isolating the O-tosyl compound obtained, adding theretoabout 10 times equivalents or so of sodium azide in excess, and allowingthe reaction in a solvent such as DMF at 50-90° C. for several hours.

Amination of compound (b-13) to compound (b-14) is normally achieved byreacting 1 equivalent of the azide compound, using 0.1 equivalent or soof a catalyst like palladium hydroxide, in the presence of a solventsuch as methanol under 1 to several atmospheric pressures of hydrogen atroom temperature for several hours.

Process 5: Production Method for the Hydrophilic Spacer Having a PartialStructure Represented by General Formula (Ic)

In each structural formula, particular groups and particular compoundsare shown in some cases, which, however, are given for exemplificationand are not to be construed as limiting. They are appropriatelyvariable, as long as they retain an equivalent function.

In the formulas, the definitions for individual symbols are as describedabove. The hydroxyl-group-protecting groups, amino-group-protectinggroups and carboxyl-group-protecting groups in the formulas are givenfor exemplification, in addition to which groups optionally chosengroups in common use in the art are used. Specifically, the same asthose described above can be mentioned as examples. It will be obviousto those skilled in the art that amino group protection, carboxyl groupdeprotection, and hydroxyl group protection and deprotection can beappropriately performed using known methods and reagents according tothe protective group used, in addition to those described in the presentspecification.

Hydroxyl group protection of compound (c-1) to compound (c-2), whenusing TBS, for example, as the protective group, is normally conductedby reacting 1 equivalent of the phenol compound, 3 equivalents or so ofa base (e.g., imidazole) and 2 equivalents or so of silyl chloride in asolvent such as DMF at room temperature for 10 hours or so.

The dehydration-condensation reaction of compound (c-2) and compound(c-4) is normally conducted by reacting 1 equivalent of the alcoholcompound and 1 equivalent of tributylphosphine in a toluene solvent atroom temperature for 1 hour or so, adding thereto 1.3 equivalents of thephenol compound and 1.3 equivalents of a condensing agent such as1,1′-azobis(N,N-dimethylformamide), and allowing the reaction at roomtemperature for several hours to overnight.

Hydroxyl group deprotection of compound (c-7) to compound (c-8) isnormally conducted by reacting 1 equivalent of the phenol-protectedcompound (e.g., silyl-protected compound) and 1.2 equivalents or so oftetrabutylammonium fluoride in a solvent such as THF at room temperaturefor 1 hour or so.

The condensation reaction of compound (c-8) and compound (c-6) isnormally conducted by reacting 1 equivalent of the phenol compound andabout 5.2 equivalents of a strong base like sodium hydride in excess atroom temperature in a solvent such as THF or DMF for 10-60 minutes orso, adding thereto 4 equivalents or so of a halide (e.g., alkylbromide), and allowing the reaction at room temperature for about 4hours or so. By this condensation reaction, compound (c-9) is obtained.

Hydroxyl group deprotection of compound (c-9) to compound (c-10) isnormally conducted by reacting 1 equivalent of the phenol-protectedcompound (e.g., trityl-protected compound) in a solvent such asmethylene chloride that contains TEA at room temperature for about 1hour or so.

Hydroxyl group protection of compound (c-10) to compound (c-11), whenusing a tert-butoxycarbonyl group, for example, as the protective group,is normally conducted by reacting 1 equivalent of the alcohol compound,about 4 equivalents of a strong base such as sodium hydride, and about 4equivalents of bromoacetic acid tert-butyl ester in a solvent such asTHF or DMF at room temperature for about 4 hours or so.

Hydroxyl group deprotection of compound (c-11) to compound (c-12) isnormally conducted by reacting 1 equivalent of a phenol-protectedcompound (e.g., benzyl-protected compound) and a catalytic amount ofpalladium hydroxide in a hydrogen gas atmosphere in a solvent such asmethanol at room temperature for about 6 hours or so.

Hydroxyl group protection of compound (c-12) to compound (c-13), whenusing Ts, for example, as the protective group, is normally conducted byreacting 1 equivalent of the alcohol compound, a catalytic amount of abase such as DMAP, and about 6 equivalents of tosyl chloride in asolvent such as pyridine at room temperature to 40° C. for about 2 hoursor so.

Azidation of compound (c-13) to compound (c-14) is conducted by reacting1 equivalent of the tosyl compound and about 15 equivalents of sodiumazide in a solvent such as DMF at about 60° C. for about 2 hours or so.

Amination of compound (c-14) to compound (c-15) andamino-group-protecting group introduction to compound (C-16) arenormally conducted by reacting 1 equivalent of the phenol-protectedcompound (benzyl-protected compound) and a catalytic amount of palladiumhydroxide in a hydrogen gas atmosphere in a solvent such as methanol atroom temperature for about 1 hour or so, adding to the amine compoundobtained (c-15) about 0.84 equivalents of 9-fluorenylmethylsuccinimidylcarbonate and about 1.5 equivalents of a base like triethylamine, andallowing the reaction in a solvent such as THF at room temperature forabout 1 hour or so.

Carboxyl group deprotection of compound (c-16) to compound (c-17) isnormally conducted by reacting 1 equivalent of the phenol-protectedcompound (e.g., t-butyl-protected compound) in an aqueous solution thatcontains TFA at room temperature for about 10 hours or so.

Process 6: Production Method for the Hydrophilic Spacer Having a PartialStructure Represented by General Formula (Id) (R₁₀=R₉=Hydrogen Atom,R₈=Hydrogen Atom, X₄=Single Bond)

In each structural formula, particular groups and particular compoundsare shown in some cases, which, however, are given for exemplificationand are not to be construed as limiting. They are appropriatelyvariable, as long as they retain an equivalent function.

In the formulas, W₈ is a hydroxyl-group-protecting group, and thedefinitions for the other symbols are as described above. As examples ofthe hydroxyl-group-protecting group, the same as those described abovecan be mentioned. Hydroxyl group deprotection is appropriately performedusing known methods and reagents according to the protective group used.

Carboxylation from compound (d-4) to compound (d-5) is normally achievedby reacting 1 equivalent of the alcohol compound with 10 equivalents ofsodium periodate, 0.4 equivalents or so of an oxidant like rutheniumchloride hydrate (III) in the presence of a solvent such as water,acetonitrile or dichloromethane at room temperature for several hours.

Process 7: Production Method (1) for the Hydrophilic Spacer Having aPartial Structure Represented by General Formula (Ie)

In each structural formula, particular groups and particular compoundsare shown in some cases, which, however, are given for exemplificationand are not to be construed as limiting. They are appropriatelyvariable, as long as they retain an equivalent function.

As examples of the hydroxyl-group-protecting group and theamino-group-protecting group, the same as those described above can bementioned. Hydroxyl group deprotection is appropriately performed usingknown methods and reagents according to the protective group used.

The carbonyl group reduction reaction of compound (e-2) to compound(e-3) is conducted by reacting 1.2 equivalents or so of a reducing agentlike NaBH₄ in a solvent such as methanol, and subsequently normallycarrying out an azide group reduction reaction Lamination) of 1equivalent of the azide compound and 0.1 equivalent or so of a catalystlike palladium hydroxide in the presence of a solvent such as methanolunder 1 to several atmospheric pressures of hydrogen at room temperaturefor several hours.

Hydroxyl group deprotection of compound (e-3) to compound (e-4) can beconducted by reacting an alkali such as 1N sodium hydroxide in a mixedsolvent of dioxane, water and the like, and subsequently protecting theamino group in the same manner as the reaction from (c-15) to (c-16).

Hydroxyl group protection of compound (e-4) to compound (e-5) can, forexample, be conducted by reacting 20 equivalents or so of TBDMS-OTf inthe presence of 2,6-Lutidine and the like.

Hydroxyl group deprotection of compound (e-5) to compound (e-6) can beconducted by allowing the reaction with 10% formic acid/dichloromethane,and subsequently oxidizing the alcohol in the same manner as thereaction from compound (d-4) to compound (d-5).

Process 8: Production Method (2) for the hydrophilic spacer having apartial structure represented General Formula (Ie)(R₁₃-R₁₆=H, R₁₁=H, R₁₂=H, r=1)

In the formulas, the definitions for individual symbols are as describedabove. The hydroxyl-group-protecting groups, amino-group-protectinggroups and carboxyl-group-protecting groups in the formulas are givenfor exemplification, in addition to which groups optionally chosengroups in common use in the art are used. Specifically, the same asthose described above can be mentioned as examples. It will be obviousto those skilled in the art that amino group protection, carboxyl groupdeprotection and hydroxyl group protection can be appropriatelyperformed using known methods and reagents according to the protectivegroup used, in addition to those described in the present specification.

Azidation from compound (e-8) to compound (e-9) is conducted by reacting1 equivalent of compound (e-8), a catalytic amount of a base such asDMAP, and about 10 equivalents of tosyl chloride in a solvent such asmethylene chloride at room temperature to 40° C. for about 2 hours toovernight to yield a tosyl derivative of compound (e-8), and reacting 1equivalent of the tosyl derivative obtained with about 15 equivalents ofsodium azide in a solvent such as DMSO at about 60-70° C. for about 5hours or so.

By aminating compound (e-9), and subsequently protecting the aminogroup, a compound having a partial structure represented by Formula (Ie)is obtained.

Usually, the amine compound is obtained by reacting 1 equivalent ofcompound (e-9) and a catalytic amount of palladium hydroxide in ahydrogen atmosphere in a solvent such as methanol or ethanol at roomtemperature for about 1-2 hours or so. Next, an amino-group-protectinggroup is introduced by reacting the amine compound obtained inaccordance with a conventional method using, for example,9-fluorenylmethylsuccinimidyl carbonate and the like, in the presence ofa base like triethylamine in a solvent such as THF.

In the present invention, in addition to the compounds mentioned aboveas hydrophilic spacers, the polymers obtained by polymerizing them canalso be used as hydrophilic spacers. For the polymerization, variousmethods in general use in the art can be employed.

Specifically, the polymerization is conducted by subjecting compoundsdescribed above to chemical reactions such as amidation,N-substitutional amidation, Schiff base formation (after Schiff baseformation, the relevant portion may be subjected to a reductionreaction), esterification, and epoxy cleavage reaction with an amine ora hydroxyl group. Although the polymerization reaction can be conductedwhile the starting monomer component is in a free state, it ispreferable, because of the ease of the subsequent purification step, toimmobilize the starting monomer component onto a metal surface and thenconduct the polymerization reaction on the metal surface. The reagentsand reaction conditions used for these reactions are according tomethods in common use in the art.

EXAMPLES

The present invention is hereinafter described in more detail by meansof the following production examples, an example and an experimentalexample, which examples, however, are not to be construed as limitingthe scope of the present invention.

Production Example 1 Synthesis of17-allyl-14-(tert-butyl-dimethyl-silanyloxy)-1-hydroxy-12-{2-[4-(7-(tert-butyl-dimethyl-silanyloxy-carbonyl)heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraone

A mixture of17-allyl-14-(tert-butyl-dimethyl-silanyloxy)-1-hydroxy-12-[2-(4-hydroxy-3-methoxy-cyclohexyl)-1-methyl-vinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraone(FK506; 138 mg, 0.15 mmol),O-mono(tert-butyl-dimethyl-silanyl)octanedioic acid (86.7 mg, 0.218mmol), dimethylaminopyridine (DMAP; 16.5 mg, 0.098 mmol),1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC.HCl;69.1 mg, 0.261 mmol) and methylene chloride (CH₂Cl₂; 1 ml) was stirredat room temperature for 1.5 hours. The reaction product was poured overan ethyl acetate-water mixed fluid and extracted. The organic phaseobtained was washed with water and brine, after which it was dried withmagnesium sulfate (MgSO₄). After the MgSO₄ was separated by filtration,concentration under reduced pressure was conducted. The residue thusobtained was purified using a silica gel column (eluted with 20% AcOEt(in n-hexane)) to yield the desired17-allyl-14-(tert-butyl-dimethyl-silanyloxy)-1-hydroxy-12-{2-[4-(7-(tert-butyl-dimethyl-silanyloxy-carbonyl)heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraone(44 mg, 24.6%).

¹H-NMR (CDCl₃) δ: −0.1-0.1 (12H, m), 0.7-2.6 (47H, m), 0.85 and 0.86(18H, s), 1.50 (3 H, s), 1.63 (3H, s), 2.75 (1H, m), 3.31 (3H, s), 3.35(3H, s), 3.39 (3H, s), 4.05 (1H, m), 3.0-4.4 (6H), 4.5-5.8 (9H, m).

Production Example 2 Synthesis of17-allyl-1,14-dihydroxy-12-{2-[4-(7-carboxy-heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraone

To a mixture of the17-allyl-14-(tert-butyl-dimethyl-silanyloxy)-1-hydroxy-12-{2-[4-(7-(tert-butyl-dimethyl-silanyloxy-carbonyl)heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraoneprepared in Production Example 1 (44 mg, 0.037 mmol) and acetonitrile(0.88 ml), 46-48% aqueous hydrogen fluoride (HF) (0.12 ml) was gentlyadded, and this was followed by overnight stirring at room temperature.The reaction product was poured over an ethyl acetate-water mixed fluidand extracted. The organic phase obtained was washed with water andbrine, after which it was dried with magnesium sulfate (MgSO₄). Afterthe MgSO₄ was separated by filtration, concentration under reducedpressure was conducted. The residue thus obtained was purified using asilica gel column (5% methanol (in chloroform)) to yield the desired17-allyl-1,14-dihydroxy-12-{2-[4-(7-carboxy-heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraone(14.2 mg, 40%).

¹H-NMR (CDCl₃) δ: 0.7-2.6 (47H, m), 1.50 (3H, s), 1.63 (3H, s), 2.75(1H, m), 3.31 (3H, s), 3.35 (3H, s), 3.39 (3H, s), 4.05 (1H, m), 3.0-4.4(6H), 4.5-5.8 (11H, m).

MS (m/z): 960 (M⁺)

Production Example 3 Synthesis (1-1) of Hydrophilic Spacer MoleculeSynthesis of2-(2-{2-[2-(2-trityloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)ethanol

Pentaethylene glycol (compound 1; 10 g, 42.0 mmol) was dissolved inpyridine (100 ml), triphenylmethyl chloride (11.6 g, 41.6 mmol) and4-dimethylaminopyridine (0.9 g, 7.4 mmol) were added at roomtemperature, and this was followed by overnight stirring at 35° C. Thiswas concentrated under reduced pressure; the residue obtained wasdissolved in chloroform, the organic phase was washed with saturatedaqueous sodium hydrogen carbonate and saturated brine, after which itwas dried with sodium sulfate. The solid was removed by cottonfiltration and washed with chloroform, and the filtrate and the washingswere combined and concentrated under reduced pressure. The residueobtained was subjected to silica gel column chromatography (KantoChemical 60N; 600 ml) with an eluent (60:1 chloroform (CHCl₃)-methanol(MeOH)) to yield the desired2-(2-{2-[2-(2-trityloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)ethanol (compound2; 10.4 g, 51.2%).

¹H-NMR (CDCl₃) δ: 2.53 (1H, t), 3.16 (2H, t), 3.49-3.63 (18H, m),7.14-7.41 (15H, m).

Production Example 4 Synthesis (1-2) of Hydrophilic Spacer MoleculeSynthesis of[2-(2-{2-[2-(2-trityloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]aceticacid

The compound 2 obtained in Production Example 3 (10.2 g, 21.2 mmol) wasdissolved in a mixed solvent of tetrahydrofuran (THF; 200 ml) and DMF(50 ml), sodium hydride (3.1 g; oily, 60 wt %) was added little bylittle at 0° C., and this was followed by stirring at room temperaturefor 30 minutes. After this was cooled to 0° C., bromoacetic acid (6.5 g,46.8 mmol) was added little by little, and this was followed by stirringat room temperature for 30 minutes. Subsequently, sodium hydride (11.6g; oily, 60 wt %) was further added little by little at roomtemperature, and this was followed by stirring at room temperature for 1hour. The reaction solution was cooled to 0° C., and water (25 ml) wasgradually added, after which the reaction solution was concentratedunder reduced pressure until the volume thereof became about 100 ml.Ethyl acetate (200 ml) and brine (100 ml) were added thereto, and 2Maqueous potassium hydrogen sulfate was added with stirring to obtain apH of 6. The organic phase was extracted and concentrated under reducedpressure at 30° C.; the residue obtained was subjected to silica gelcolumn chromatography (Kanto Chemical 60N; 400 ml) with an eluent (85:15CHCl₃-MeOH) to yield a crude product of the desired[2-(2-{2-[2-(2-trityloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]aceticacid (compound 3) (12.4 g).

¹H-NMR (CDCl₃) δ: 3.34 (2H, t), 3.76-3.84 (20H, m), 4.13 (2H, s),7.30-7.83 (15H, m).

Production Example 5 Synthesis (1-3) of Hydrophilic Spacer MoleculeSynthesis of[2-(2-{2-[2-(2-trityloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]aceticacid benzyl ester

The crude product of compound 3 obtained in Production Example 4 (12.4g) was dissolved in methylene chloride (100 ml), and4-dimethylaminopyridine (0.29 g, 2.4 mmol) and benzyl alcohol (3.1 ml,30.0 mmol) were added. This was cooled to 0° C., water-solublecarbodiimide (N-ethyl-N′-(3′-dimethylaminopropyl)carbodiimide; WSC; 4.5g, 23.5 mmol) was added, and this was followed by overnight stirring atroom temperature. The reaction solution was extracted with chloroform,and the organic phase was washed with saturated aqueous sodium hydrogencarbonate and saturated brine, after which it was dried with sodiumsulfate. The solid was removed by cotton filtration and washed withchloroform, and the filtrate and the washings were combined andconcentrated under reduced pressure. The residue obtained was subjectedto silica gel column chromatography (Kanto Chemical 60N; 600 ml) with aneluent (1:1 ethyl acetate-hexane) to yield the desired[2-(2-{2-[2-(2-trityloxy-ethoxy)-ethoxy]-ethoxy}-ethoxy -ethoxy]aceticacid benzyl ester (compound 4; 12.0 g, 90.1%, 2 steps).

¹H-NMR (CDCl₃) δ: 3.16 (2H, t), 3.55-3.65 (20H, m), 4.11 (2H, s), 5.11(2H, s), 7.15-7.40 (20H, m).

Production Example 6 Synthesis (1-4) of Hydrophilic Spacer MoleculeSynthesis of[2-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]aceticacid benzyl ester

The compound 4 obtained in Production Example 5 (12.0 g) was dissolvedin a 5% solution of trifluoroacetic acid in methylene chloride (150 ml),water (10 ml) was added at 0° C., and this was followed by stirring at0° C. for 20 minutes. The reaction solution was poured over saturatedaqueous sodium hydrogen carbonate, extracted, and dried with sodiumsulfate. The solid was removed by cotton filtration and washed withchloroform, and the filtrate and the washings were combined andconcentrated under reduced pressure. The residue obtained was subjectedto silica gel column chromatography (Kanto Chemical 60N; 400 ml) with aneluent (1000:15 CHCl₃-MeOH) to yield the desired[2-(2-{2-[2-(2-hydroxy-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]aceticacid benzyl ester (compound 5; 7.0 g, 95%).

¹H-NMR (CDCl₃) δ: 2.80 (1H, t), 3.62-3.76 (20H, m), 4.22 (2H, s), 5.20(2H, s), 7.36-7.41 (5H, m).

Production Example 7 Synthesis (1-5) of Hydrophilic Spacer MoleculeSynthesis of[2-(2-{2-[2-(2-azido-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]acetic acidbenzyl ester

The compound 5 obtained in Production Example 6 (7.0 g, 18.1 mmol) and4-dimethylaminopyridine (0.4 g, 3.3 mmol) were dissolved in pyridine (45ml), and this solution was cooled to 0° C. p-Toluenesulfonyl chloride(5.2 g, 27.2 mmol) was added thereto, this was followed by overnightstirring at room temperature, p-toluenesulfonyl chloride (3.1 g, 16.2mmol) and 4-dimethylaminopyridine (120 mg, 0.98 mmol) were furtheradded, and this was followed by stirring at 30° C. for 2 hours. Thereaction solution was cooled to 0° C., water (3 ml) was added,concentration under reduced pressure was conducted, the residue obtainedwas dissolved in ethyl acetate, and the organic phase was washed withsaturated aqueous sodium hydrogen carbonate and saturated brine, afterwhich it was dried with sodium sulfate. The solid was removed by cottonfiltration and washed with ethyl acetate, and the filtrate and thewashings were combined and concentrated under reduced pressure. Theresidue obtained was dissolved in DMF (50 ml), sodium azide (11.8 g,0.18 mol) was added, and this was followed by stirring at 60° C. for 1hour. The reaction solution was extracted with ethyl acetate, and theorganic phase was washed with saturated aqueous sodium hydrogencarbonate and saturated brine, after which it was dried with sodiumsulfate. The solid was removed by cotton filtration and washed withethyl acetate, and the filtrate and the washings were combined andconcentrated under reduced pressure. The residue obtained was subjectedto silica gel column chromatography (Kanto Chemical 60N; 250 ml) with aneluent (3:1 ethyl acetate-hexane) to yield the desired[2-(2-{2-[2-(2-azido-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]acetic acidbenzyl ester (compound 6; 3.3 g, 44.3%).

¹H-NMR (CDCl₃) δ: 3.31 (2H, t), 3.54-3.87 (20H, m), 4.13 (2H, s), 5.12(2H, s), 7.20-7.30 (5H, m).

Production Example 8 Synthesis (1-6) of Hydrophilic Spacer MoleculeSynthesis of[2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]acetic acid

The compound 6 obtained in Production Example 7 (1.94 g, 4.72 mmol) wasdissolved in methanol (50 ml), 10% Pd—C (500 mg) was added, andcatalytic hydrogenation was conducted at room temperature for 2.5 hours.The solid was removed by Celite filtration and washed with methanol, andthe filtrate and the washings were combined and concentrated underreduced pressure to yield the desired[2-(2-{2-[2-(2-amino-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]acetic acid(compound 7; 1.4 g, quantitative).

MS (m/z): 296 (M⁺)

Production Example 9 Synthesis (1-7) of Hydrophilic Spacer MoleculeSynthesis of{2-[2-(2-{2-[2-(9H-fluoren-9-yl-methoxycarbonylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}aceticacid

The compound 7 obtained in Production Example 8 (1.25 g, 4.23 mmol) wasdissolved in 10% aqueous sodium carbonate (14 ml),9-fluorenylmethylsuccinimidyl carbonate (2.15 g, 6.37 mmol) insuspension in dimethoxyethane (14 ml) was added drop by drop at roomtemperature, and this was followed by overnight stirring at roomtemperature. The solid was separated by filtration with Celite, afterwhich it was washed with chloroform. The filtrate and the washings werecombined and extracted with chloroform, and the organic phase was washedwith 2M aqueous sodium hydrogen sulfate and saturated brine and driedwith sodium sulfate. The solid was removed by cotton filtration andwashed with chloroform, and the filtrate and the washings were combinedand concentrated under reduced pressure. The residue obtained wassubjected to silica gel column chromatography (Kanto Chemical 60N; 150ml) with an eluent (1000:7 CHCl₃-MeOH) to yield the desired{2-[2-(2-{2-[2-(9H-fluoren-9-yl-methoxycarbonylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}aceticacid (compound 8; 1.38 g, 63.0%).

¹H-NMR (CDCl₃) δ: 3.34 (2H, t), 3.50-3.71 (18H, m), 4.05 (2H, s), 4.12(1H, t), 4.33 (2H, d), 5.57 (1H, s), 7.22-7.95 (8H, m).

Production Example 10 Synthesis of Gold Film Bearing Hydrophilic Spacer:Gold Film Bearing Hexaethylene Glycol Derivative (Kojundo ChemicalLaboratory Co., Ltd.; Pure Gold, Purity 99.9% up, Shape 10 mm×10 mm×0.01mm (Thickness))

A gold film (about 1 cm²) was immersed in a Piranha solution (30%hydrogen peroxide:con. sulfuric acid=1:4 mixed solution) for severalhours and washed with milli Q water (water filtered by pure waterproduction apparatus of Millipore) and ethanol. This was immersedovernight in a 1.5 mM ethanol solution (0.5 ml) of(6-mercapto-hexyl)-carbamic acid 9H-fluoren-9-yl-methyl ester to formSAM on the gold thin film. After completion of the reaction, the goldfilm was sufficiently washed with ethanol and acetonitrile, and thepresence of about 250 Pmol of amine on the gold film was confirmed bythe method described in Production Example 12.

The gold film was sufficiently washed with acetonitrile,{2-[2-(2-{2-[2-(9H-fluoren-9-yl-methoxycarbonylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}aceticacid (compound 8 obtained in Production Example 9; 12.5 mg, 0.024 mmol)dissolved in acetonitrile (0.25 ml) was added,benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP; 13 mg, 0.025 mmol) dissolved in acetonitrile (0.25 ml), andN,N-diisopropylethylamine (8.9 μl, 0.50 mmol) were further added, andthe mixture was shaken overnight at room temperature. The reactionmixture was removed, and the gold film was washed with acetonitrile, andallowed to react overnight under the same conditions. After completionof the reaction, the gold film was sufficiently washed withacetonitrile, acetic acid (0.3 μl, 0.005 mmol) dissolved in acetonitrile(0.25 ml) was added, benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate (PyBOP; 2.6 mg, 0.005 mmol) dissolved inacetonitrile (0.25 ml), and N,N-diisopropylethylamine (1.7 μl, 0.010mmol) were further added, and the mixture was shaken at room temperaturefor 5 hr. The gold film was sufficiently washed with acetonitrile andthe condensation rate was determined (about 90%) by the method describedin Production Example 12. In this way, a gold film bearing ahexaethylene glycol derivative as a hydrophilic spacer via alkanethiolderived from SAM was obtained (gold film bearing hexaethylene glycolderivative).

Production Example 11 Synthesis of Gold Film Bearing FK506Derivative-Bound Hydrophilic Spacer (Gold Film+(PEG)₁—FK506)

The gold film bearing a hexaethylene glycol derivative obtained inProduction Example 10, and a mixture of17-allyl-1,14-dihydroxy-12-{2-[4-(7-carboxy-heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraone(4.8 mg, 0.005 mmol) prepared in Production Example 2, EDC.HCl (1.0 mg,0.005 mmol), 1-hydroxybenzotriazole (HOBt; 0.7 mg, 0.005 mmol) anddimethylformamide (DMF; 0.5 ml) were stirred overnight at roomtemperature. After completion of the reaction, the gold film wassufficiently washed with DMF, immersed in a mixture of acetic acid (0.3μl, 0.005 mmol) dissolved in DMF (0.25 ml), EDC.HCl (1.0 mg, 0.005mmol), DMF (0.5 ml) containing HOBt (0.7 mg, 0.005 mmol) dissolvedtherein, and the mixture was stirred overnight at room temperature. Thegold film was sufficiently washed with dimethylformamide (DMF) andacetonitrile to give a FK506-bound gold film bearing a hydrophilicspacer [gold film+(PEG)₁—FK506].

The number of HBA of the hydrophilic spacer intervening between the goldfilm and FK506 is 7, and the number of HBD thereof is 1. However, thenumber derived from alkanethiol moiety, which is derived from SAM, andgroup introduced into FK506 in advance are not counted in.

Production Example 12 Quantitation of Immobilization Amount of LowMolecular on Gold Thin Film by Quantitation of Fluorene Derivative

In Production Example 10, the 1.5 mM ethanol solution of(6-mercapto-hexyl)-carbamic acid 9H-fluoren-9-yl-methyl ester, in whichthe gold thin film was immersed overnight, was removed and the gold filmwas sufficiently washed with ethanol and acetonitrile, immersed in anacetonitrile solution containing 1 mL of 20% piperidine, and the mixturewas shaken for 30 min. The acetonitrile solution was recovered, and thegold thin film was washed with 1 ml of acetonitrile. The recoveredacetonitrile solution and the acetonitrile solution used for washing thegold thin film were combined and concentrated under reduced pressure.The residue was vacuum dried at 50° C. for 1 hr. After allowing to coolto room temperature, the fluorene derivative attached to the inside ofthe container was dissolved with 100 μL of acetonitrile, and 100 μL ofmilli Q water was further added. The solution was filtered, subjected tomass analysis with LC/MS of a fluorene derivative generated from Fmocgroup by deprotection, and the fluorene derivative was quantitated fromthe obtained peak (M+1; 264) area. The amino group generated bydeprotection was condensed with the hydrophilic spacer with an Fmocgroup, which was obtained in Production Example 9, and deprotection with20% piperidine and mass analysis of the produced fluorene derivativewere performed, based on which the binding amount of hydrophilic spaceronto the gold thin film was determined.

Reference Example 1 Synthesis of FK506 Derivative-Bound (AlkanethiolDirectly Bonded/No Hydrophilic Spacer) Gold Film

A gold film was treated with (6-mercapto-hexyl)-carbamic acid9H-fluoren-9-yl-methyl ester according to the method described inProduction Example 10 to immobilize alkanethiol on the gold film, and17-allyl-1,14-dihydroxy-12-{2-[4-(7-carboxy-heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraonewas introduced thereinto by a method according to the description ofProduction Example 11.

Reference Example 2 Synthesis of Gold Film Bearing FK506Derivative-Bound Dextran (Gold Film+Dextran—FK506)

A gold film (Kojundo Chemical Laboratory Co., Ltd.; pure gold, purity99.9% up, shape 10 mm×10 mm×0.01 mm (thickness)) (about 1 cm²) immersedin a Piranha solution (30% hydrogen peroxide: conc. sulfuric acid=1:4mixed solution) for several hours was washed with milli Q water andethanol. Using the gold film and according to the method described in areference (J. Chem. Soc., Chem. Commun., 1526-1528, 1990), a gold filmwith carboxymethyldextran (CM-Dextran) was prepared. The obtained goldfilm with carboxymethyldextran and a mixture of17-allyl-1,14-dihydroxy-12-{2-[4-(7-carboxy-heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraone(9.6 mg, 0.01 mmol) prepared in Production Example 2, EDC.HCl (1.92 mg,0.01 mol), HOBt (1.33 mg, 0.01 mmol), triethylamine (3.03 mg, 0.03 mol)and milli Q water (1 ml) were stirred overnight at room temperature.After completion of the reaction, the gold film was sufficiently washedwith milli Q water, and immersed in a mixture of acetic acid (0.57 μl,0.01 mmol), EDC.HCl (1.92 mg, 0.01 mmol), milli Q water (1 ml)containing HOBt (1.33 mg, 0.01 mmol) dissolved therein, and the mixturewas stirred overnight at room temperature. The gold film wassufficiently washed with milli Q water to give a gold film bearingFK506-bound dextran [gold film+dextran—FK506].

Production Example 13 Synthesis of BIACORE Sensor Chip BearingHydrophilic Spacer: Sensor Chip (BIACORE; SIA Sensor Chip) BearingHexaethylene Glycol Derivative

A sensor chip (BIACORE; SIA sensor chip) was immersed in a Piranhasolution (30% hydrogen peroxide:con. sulfuric acid=1:4 mixed solution)for several hours and washed with milli Q water and ethanol. This wasimmersed overnight in a 1.5 mM ethanol solution (0.5 ml) of(6-mercapto-hexyl)-carbamic acid 9H-fluoren-9-yl-methyl ester to formSAM on the gold surface of the sensor chip. After completion of thereaction, the sensor chip was sufficiently washed with ethanol andacetonitrile, a mixed solution (1 ml) of piperidine/acetonitrile (1/4)was added and the mixture was shaken at room temperature for 30 min. Thesensor chip was sufficiently washed with acetonitrile,{2-[2-(2-{2-[2-(9H-fluoren-9-yl-methoxycarbonylamino)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}aceticacid (compound 8 obtained in Production Example 9; 12.5 mg, 0.024 mmol)dissolved in acetonitrile (0.25 ml) was added,benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP; 13 mg, 0.025 mmol) dissolved in acetonitrile (0.25 ml), andN,N-diisopropylethylamine (8.9 μl, 0.50 mmol) were further added, andthe mixture was shaken overnight at room temperature. The reactionmixture was removed, and the sensor chip was washed with acetonitrile,and allowed to react overnight under the same conditions. Aftercompletion of the reaction, the sensor chip was sufficiently washed withacetonitrile and immersed in a mixed solution of acetic acid (0.3 μl,0.005 mmol) dissolved in acetonitrile (0.25 ml),benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP; 2.6 mg, 0.005 mmol) dissolved in acetonitrile (0.25 ml), andN,N-diisopropylethylamine (1.7 μl, 0.010 mmol), and the mixture was isshaken at room temperature for 5 hr. The sensor chip was sufficientlywashed with acetonitrile and treated with a mixed solution (1 ml) ofpiperidine/acetonitrile (1/4) as mentioned above. In this way, a BIACOREsensor chip bearing a hydrophilic spacer was obtained.

Production Example 14 Synthesis of Sensor Chip Bearing FK506Derivative-Bound Hydrophilic Spacer (Sensor Chip+(PEG)₁—FK506)

The sensor chip bearing a hexaethylene glycol derivative obtained inProduction Example 13, and a mixture of17-allyl-1,14-dihydroxy-12-{2-[4-(7-carboxy-heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraone(4.8 mg, 0.005 mmol) prepared in Production Example 2, EDC.HCl (1.0 mg,0.005 mmol), HOBt (0.7 mg, 0.005 mmol) and DMF (0.5 ml) were stirredovernight at room temperature. After completion of the reaction, thesensor chip was sufficiently washed with DMF, immersed in a mixture ofacetic acid (0.3 μl, 0.005 mmol) dissolved in DMF (0.25 ml), EDC.HCl(1.0 mg, 0.005 mmol), DMF (0.5 ml) containing HOBt (0.7 mg, 0.005 mmol)dissolved therein, and the mixture was stirred overnight at roomtemperature. The sensor chip was sufficiently washed with DMF andacetonitrile to give a FK506-bound sensor chip bearing a hydrophilicspacer [sensor chip+(PEG)₁—FK506]. The number of HBA of the hydrophilicspacer intervening between the sensor chip having gold surface and FK506is 7, and the number of HBD thereof is 1. However, the number derivedfrom alkanethiol moiety which is derived from SAM, and group introducedinto FK506 in advance are not counted in.

Reference Example 3 Synthesis of FK506 Derivative-Bound (AlkanethiolDirectly Bonded/No Hydrophilic Spacer) Sensor Chip

A sensor chip was treated with (6-mercapto-hexyl)-carbamic acid9H-fluoren-9-yl-methyl ester according to the method described inProduction Example 10 to immobilize alkanethiol on the sensor chip, and17-allyl-1,14-dihydroxy-12-{2-[4-(7-carboxy-heptanoyl-oxy)-3-methoxy-cyclohexyl]-1-methyl-vinyl}-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-aza-tricyclo[22.3.1.0^(4,9)]octacos-18-ene-2,3,10,16-tetraonewas introduced thereinto by a method according to the description ofProduction Example 11.

Example 1 (1) Preparation of Lysate

The rat brain (2.2 g) was mixed in a mixed fluid A (0.25M sucrose, 25 mMTris buffer (pH 7.4), 22 ml) and prepared as a homogenate, which wasthen centrifuged at 9500 rpm for 10 minutes. The centrifugal supernatantwas collected and further centrifuged at 50000 rpm for 30 minutes. Thesupernatant thus obtained was used as the lysate. Note that allexperiments were conducted at 4° C. or on ice.

(2) Binding Experiments

Using the above-mentioned various FK506-bound gold film (FK506 boundgold foils), a binding experiment to lysate was performed by thefollowing steps. The lysate was diluted with mixed fluid A to ½ beforeuse. Each one sheet (10 mm×10 mm×0.01 mm (thickness)) of variousFK506-bound gold film was used.

As the FK506-bound gold film, a gold film bearing FK506 derivative-boundhydrophilic spacer of Production Example 11, into which a hexaethyleneglycol derivative had been introduced, was used. As a ComparativeExample, a gold film bearing FK506 (no hydrophilic spacer) of ReferenceExample 1 or a gold film bearing FK506 (dextran spacer) of ReferenceExample 2 was used.

The FK506-bound gold film and a lysate (1 ml) were gently shakenovernight at 4° C. Then, the supernatant was removed, and the resultingFK506-bound gold film was sufficiently washed 3 times with mixed fluidA, whereby each FK506-bound gold film was sufficiently washed.

To the FK506-bound gold film thus obtained, 25 μl of a loading bufferfor SDS-PAGE (Nacalai cat. NO=30566-22, sample buffer solution forelectrophoresis with 2-ME (2-mercaptoethanol) (2×) for SDS PAGE) wasadded and pipetted. The sample fluid thus obtained was separated using acommercially available SDS gel (BioRad readyGel J, 15% SDS, cat.NO=161-J341), and the SDS gel was analyzed (FIG. 1). As a result, theone shown in FIG. 1, lane 4, with an introduced hydrophilic spacershowed a decrease or disappearance of the band intensity considered tobe based on nonspecific interactions, and intensifying of the band(FKBP12) intensity considered to be based on specific interactions, ascompared to the one shown in lane 3, which was free of a hydrophilicspacer. The results indicate that the introduction of a hydrophilicspacer suppressed nonspecific interactions and intensified specificinteractions.

Example 2 (1) Preparation of Lysate

Performed according to Example 1.

(2) Binding Experiment

Using the above-mentioned various FK506-bound sensor chips, a bindingexperiment to lysate was performed by the following steps. The lysatewas diluted with mixed fluid A to ½ before use. Each one sheet ofvarious FK506-bound sensor chips was used.

As the FK506-bound sensor chip, sensor chip bearing FK506-boundhydrophilic spacer of Production Example 14, into which a hexaethyleneglycol derivative had been introduced, was used. As a ComparativeExample, an FK506-bound sensor chip of Reference Example 3 was used.

The FK506-bound sensor chip and a lysate (1 ml) were gently shakenovernight at 4° C. Then, the supernatant was removed, and the resultingFK506-bound sensor chip was sufficiently washed 3 times with mixed fluidA, whereby each FK506-bound sensor chip surface was sufficiently washed.

To the gold surface of the FK506-bound sensor chip thus obtained, 25 μlof a loading buffer for SDS PAGE (Nacalai cat. NO=30566-22, samplebuffer solution for electrophoresis with 2-ME (2-mercaptoethanol) (2×)for SDS PAGE) was added and pipetted. The sample fluid thus obtained wasseparated using a commercially available SDS gel (BioRad readyGel J, 15%SDS, cat. NO=161-J341), and the SDS gel was analyzed. As a result, theone with an introduced hydrophilic spacer showed a decrease ordisappearance of the band intensity considered to be based onnonspecific interactions, and intensifying of the band (specifically, aband corresponding to FKBP12) intensity considered to be based onspecific interactions. The results indicate that the introduction of ahydrophilic spacer suppressed nonspecific interactions and intensifiedspecific interactions.

INDUSTRIAL APPLICABILITY

Introduction of a hydrophilic spacer in between a metal surface and aligand to be the examination target, during immobilization of the ligandon the surface of metal as a solid phase carrier enables reduction ofhydrophobic property of the metal surface, and suppression ofnonspecific intermolecular interactions. Simultaneously, specificintermolecular interactions can be intensified.

In a study including measurement of interactions of low molecule—lowmolecule, low molecule—high molecule, high molecule—high molecule, orpurification of an object target based on the interaction, it ispossible to artificially suppress nonspecific interactions or intensifyspecific interactions, by the technique of the present invention. To bespecific, the present technique facilitates a study includingimmobilizing one molecule of low molecule—high molecule, lowmolecule—low molecule, high molecule—high molecule on a solid phasecarrier and measuring interactions thereof, or purifying an objecttarget based on the interaction. Such achievement is widely applicableto life science in general, particularly drug discovery research,post-genomic research, proteomics, chemical genomics, chemicalproteomics and the like.

1. A method of suppressing a nonspecific interaction between a ligandand/or a metal surface and a molecule other than a target molecule,which comprises, in a process of immobilizing the ligand onto the metalsurface and analyzing a specific interaction on the metal surfacebetween the ligand and the target molecule thereof, a treatment toreduce the hydrophobic property of the metal surface.
 2. A method ofintensifying a specific interaction between a ligand and a targetmolecule, which comprises, in a process of immobilizing the ligand ontoa metal surface and analyzing a specific interaction on the metalsurface between the ligand and the target molecule of the ligand, atreatment to reduce the hydrophobic property of the metal surface.
 3. Amethod of suppressing a nonspecific interaction between a ligand and/ora metal surface and a molecule other than a target molecule andintensifying a specific interaction between the ligand and the targetmolecule, which comprises, in a process of immobilizing the ligand ontothe metal surface and analyzing a specific interaction on the metalsurface between the ligand and the target molecule of the ligand, atreatment to reduce the hydrophobic property of the metal surface.
 4. Amethod of suppressing a nonspecific interaction between a ligand and/ora metal surface and a molecule other than a target molecule, whichcomprises, in a process of immobilizing the ligand onto the metalsurface and selecting a target molecule using a specific interaction onthe metal surface between the ligand and the target molecule thereof, atreatment to reduce the hydrophobic property of the metal surface.
 5. Amethod of intensifying a specific interaction between a ligand and atarget molecule, which comprises, in a process of immobilizing theligand onto the metal surface and selecting the target molecule using aspecific interaction on the metal surface between the ligand and thetarget molecule thereof, a treatment to reduce the hydrophobic propertyof the metal surface.
 6. A method of suppressing a nonspecificinteraction between a ligand and/or a metal surface and a molecule otherthan a target molecule and intensifying a specific interaction betweenthe ligand and the target molecule, which comprises, in a process ofimmobilizing the ligand onto the metal surface and selecting the targetmolecule using a specific interaction on the metal surface between theligand and the target molecule thereof, a treatment to reduce thehydrophobic property of the metal surface.
 7. The method of claim 1,wherein the treatment to reduce the hydrophobic property of the metalsurface is to introduce, at the time of immobilization of the ligandonto the metal surface, a hydrophilic spacer therebetween.
 8. The methodof claim 7, wherein the hydrophilic spacer has at least any of thefollowing characteristics while in a state bound to the metal surfaceand the ligand: (i) the number of hydrogen bond acceptor is 6 or more,(ii) the number of hydrogen bond donor is 5 or more, (iii) the totalnumber of hydrogen bond acceptor and hydrogen bond donor is 9 or more.9. The method of claim 8, wherein said hydrophilic spacer further hasone or more carbonyl groups in the molecule thereof.
 10. The method ofclaim 8, further characterized in that said hydrophilic spacer does nothave a functional group that becomes positively or negatively charged inan aqueous solution.
 11. A method of immobilizing a ligand onto a metalsurface and analyzing a specific interaction on the metal surfacebetween the ligand and a target molecule thereof which comprisessuppressing a nonspecific interaction between the ligand and/or themetal surface and a molecule other than the target molecule by atreatment to reduce the hydrophobic property of the metal surface.
 12. Amethod of immobilizing a ligand onto a metal surface and analyzing aspecific interaction on the metal surface between the ligand and atarget molecule thereof, which comprises intensifying a specificinteraction between the ligand and the target molecule by a treatment toreduce the hydrophobic property of the metal surface.
 13. A method ofimmobilizing a ligand onto a metal surface and analyzing a specificinteraction on the metal surface between the ligand and a targetmolecule thereof, which comprises suppressing a nonspecific interactionbetween the ligand and/or the metal surface and a molecule other thanthe target molecule and intensifying a specific interaction between theligand and the target molecule, by a treatment to reduce the hydrophobicproperty of the metal surface.
 14. A method of immobilizing a ligandonto a metal surface, and selecting a target molecule using a specificinteraction on the metal surface between the ligand and the targetmolecule thereof, which comprises suppressing a nonspecific interactionbetween the ligand and/or the metal surface and a molecule other thanthe target molecule by a treatment to reduce the hydrophobic property ofthe metal surface.
 15. A method of immobilizing a ligand onto a metalsurface, and selecting a target molecule using a specific interaction onthe metal surface between the ligand and the target molecule thereof,which comprises intensifying a specific interaction between the ligandand the target molecule by a treatment to reduce the hydrophobicproperty of the metal surface.
 16. A method of immobilizing a ligandonto a metal surface, and selecting a target molecule using a specificinteraction on the metal surface between the ligand and the targetmolecule thereof, which comprises suppressing a nonspecific interactionbetween the ligand and/or the metal surface and a molecule other thanthe target molecule and intensifying a specific interaction between theligand and the target molecule, by a treatment to reduce the hydrophobicproperty of the metal surface.
 17. The method of claim 11, wherein thetreatment to reduce the hydrophobic property of the metal surface is tointroduce, at the time of immobilization of the ligand onto the metalsurface, a hydrophilic spacer therebetween.
 18. The method of claim 17,wherein the hydrophilic spacer has at least any of the followingcharacteristics while in a state bound to the metal surface and theligand: (i) the number of hydrogen bond acceptor is 6 or more, (ii) thenumber of hydrogen bond donor is 5 or more, (iii) the total number ofhydrogen bond acceptor and hydrogen bond donor is 9 or more.
 19. Themethod of claim 18, wherein said hydrophilic spacer further has one ormore carbonyl groups in the molecule thereof.
 20. The method of claim18, further characterized in that said hydrophilic spacer does not havea functional group that becomes positively or negatively charged in anaqueous solution.
 21. A screening method for a target molecule having aspecific interaction with a ligand, comprising at least the followingsteps: (i) immobilizing the ligand onto a metal surface via ahydrophilic spacer, (ii) contacting a sample that contains or does notcontain the target molecule with the metal surface with the ligandimmobilized thereon obtained in (i) above, (iii) identifying andanalyzing a molecule that has exhibited or has not exhibited a specificinteraction with the ligand, and (iv) judging a molecule that exhibits aspecific interaction with the ligand as the target molecule on the basisof the analytical results obtained in (iii) above.
 22. The method ofclaim 21, wherein the hydrophilic spacer has at least any of thefollowing characteristics while in a state bound to the metal surfaceand the ligand: (i) the number of hydrogen bond acceptor is 6 or more,(ii) the number of hydrogen bond donor is 5 or more, (iii) the totalnumber of hydrogen bond acceptor and hydrogen bond donor is 9 or more.23. The method of claim 22, wherein said hydrophilic spacer further hasone or more carbonyl groups in the molecule thereof.
 24. The method ofclaim 22, further characterized in that said hydrophilic spacer does nothave a functional group that becomes positively or negatively charged inan aqueous solution.
 25. The method of claim 7, wherein the hydrophilicspacer has at least one partial structure represented by any one formulaselected from the group consisting of Formulas (Ia)-(Ie) below:

(In Formula (Ia), A is an appropriate joining group, X₁-X₃ are the sameor different and each is a single bond or a methylene group that may besubstituted by a linear or branched alkyl group having 1-3 carbon atoms,R₁-R₇ are the same or different and each is a hydrogen atom, a linear orbranched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup, m is an integer of 0-2, m′ is an integer of 0-10, m″ is aninteger of 0-2, when a plurality of R₃-R₇ units exist, they may be thesame or different, when a plurality of X₃ units exist, they may be arethe same or different; in Formula (Ib), n and n′ are the same ordifferent and each is an integer of 1-1000; in Formula (Ic), p, p′ andp″ are the same or different and each is an integer of 1-1000; inFormula (Id), X₄ is a single bond or a methylene group that may besubstituted by a linear or branched alkyl group having 1-3 carbon atoms,R₈-R₁₀ are the same or different and each is a hydrogen atom, a linearor branched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup, q is an integer of 1-7, when a plurality of R₈ units exist, theymay be the same or different, when a plurality of X₄ units exist, theymay be the same or different; in Formula (Ie), R₁₁-R₁₆ are the same ordifferent and each is a hydrogen atom, a linear or branched alkyl grouphaving 1-3 carbon atoms, —CH₂OH or a hydroxyl group, r is an integer of1-10, r′ is an integer of 1-50, when a plurality of R₁₁-R₁₆ units exist,they may be the same or different).
 26. The method of claim 25, whereinthe hydrophilic spacer has two or more partial structures represented byany one formula selected from the group consisting of Formulas(Ia)-(Ie).
 27. A solid phase carrier with a ligand immobilized thereon,which is a metal and has a hydrophilic spacer between the metal and theligand.
 28. The solid phase carrier of claim 27, wherein the hydrophilicspacer has at least one partial structure represented by any one formulaselected from the group consisting of Formulas (Ia)-(Ie) below:

(In Formula (Ia), A is an appropriate joining group, X₁-X₃ are the sameor different and each is a single bond or a methylene group that may besubstituted by a linear or branched alkyl group having 1-3 carbon atoms,R₁-R₇ are the same or different and each is a hydrogen atom, a linear orbranched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup, m is an integer of 0-2, m′ is an integer of 0-10, m″ is aninteger of 0-2, when a plurality of R₃-R₇ units exist, they may be thesame or different, when a plurality of X₃ units exist, they may be arethe same or different; in Formula (Ib), n and n′ are the same ordifferent and each is an integer of 1-1000; in Formula (Ic), p, p′ andp″ are the same or different and each is an integer of 1-1000; inFormula (Id), X₄ is a single bond or a methylene group that may besubstituted by a linear or branched alkyl group having 1-3 carbon atoms,R₈-R₁₀ are the same or different and each is a hydrogen atom, a linearor branched alkyl group having 1-3 carbon atoms, —CH₂OH or a hydroxylgroup, q is an integer of 1-7, when a plurality of R₈ units exist, theymay be the same or different, when a plurality of X₄ units exist, theymay be the same or different; in Formula (Ie), R₁₁-R₁₆ are the same ordifferent and each is a hydrogen atom, a linear or branched alkyl grouphaving 1-3 carbon atoms, —CH₂OH or a hydroxyl group, r is an integer of1-10, r′ is an integer of 1-50, when a plurality of R₁₁-R₁₆ units exist,they may be the same or different).
 29. The solid phase carrier of claim27, wherein the metal is gold.
 30. A method of confirming introductionof a hydrophilic spacer between a ligand and a metal surface, whichcomprises, in a step of introducing the hydrophilic spacer between themduring immobilization of the ligand onto the metal surface, detecting aleaving group produced by elimination of a protecting group derived fromthe hydrophilic spacer.
 31. The method of claim 30, wherein the leavinggroup is detected by mass analysis.
 32. The method of claim 30, whereinthe protecting group is a 9-fluorenylmethyloxycarbonyl group.